Matei et al. Parasites & Vectors (2018) 11:193 https://doi.org/10.1186/s13071-018-2791-y

SHORT REPORT Open Access New records for Anaplasma phagocytophilum infection in small mammal species Ioana Adriana Matei1,2*, Gianluca D’Amico1, Angela Monica Ionică1, Zsuzsa Kalmár1, Alexandra Corduneanu1, Attila D. Sándor1, Nicodim Fiţ2, Liviu Bogdan3,Călin M. Gherman1 and Andrei Daniel Mihalca1

Abstract Background: -borne diseases pose a major threat in public health. The epidemiological dynamics of these diseases depends on the tick vector species and their hosts, as well as the geographical distribution and ecology of both. Among many possible hosts for , small mammals have a major role in the development of immature stages of several tick species. Small mammals are also important reservoir hosts for several pathogenic agents and possible reservoirs for Anaplasma phagocytophilum. In this context, the aim of our study was to evaluate the prevalence of A. phagocytophilum in small mammal species in Romania. Results: A total of 791 small mammals of 31 species were tested by PCR, targeting the rrs gene for detection of A. phagocytophilum DNA. Positive results were obtained in 20 small mammals: five Apodemus flavicollis (6.49%), three Sorex araneus (9.09%), three A. uralensis (4.84%), two A. sylvaticus (3.92%), and one of each Spermophilus cittelus (7.14%), Microtus agrestis (3.85%), Sorex minutus (3.85%), Muscardinus avellanarius (3.13%), Crocidura suaveolens (2.44%), Mus spicilegus (2%) and M. arvalis (1.75%). Conclusions: Eleven small mammal species were found to be carriers of A. phagocytophilum, suggesting a possible involvement of these species in its epidemiology. To our knowledge, this is the first report of A. phagocytophilum in S. minutus, C. suaveolens, M. spicilegus, M. avellanarius and S. citellus. Keywords: Anaplasma phagocytophilum, Small mammals, Prevalence, Romania

Background important factors of disease risk [4], playing an import- Small mammals (Orders Rodentia and Eulipotyphla) ant role in the ecology of ticks and tick-borne diseases. represent a very diverse group of terrestrial vertebrates, Small mammals are important hosts for several tick with a worldwide distribution and usually represented by species, having an essential role in the development of large populations [1, 2]. They are highly adapted for immature stages of hard ticks, and also being essential various types of habitat, including urbanized areas, being maintenance hosts for the immature stages of a link between wild and anthropomorphic ecosystems ricinus [1, 5]. For instance, in a study focused on rodent- through the frequent movement of these and tick associations in Romania, a high prevalence (over their ticks between dwellings and natural envi- 50%) of tick parasitism was found especially in Microtus ronments [3]. Fluctuations in their densities are very arvalis, Apodemus uralensis, Apodemus flavicollis and Myodes glareolus [6]. Among the tick species found on small mammals in the Palaearctic, the genus Ixodes is * Correspondence: [email protected] the most well-represented: I. angustus, I. apronophorus, 1Department of Parasitology and Parasitic Diseases, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, I. crenulatus, I. hexagonus, I. laguri, I. nipponensis, I. Cluj-Napoca, Romania occultus, I. pomerantzevi, I. redikorzevi/I. acuminatus, I. 2 Department of Microbiology, Immunology and Epidemiology, Faculty of ricinus and I. trianguliceps (reviewed in [1]). Even if Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania some of these ticks are endophilic (nidicolous) (e.g. I. Full list of author information is available at the end of the article

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trianguliceps and I. acuminatus) and normally do not Polymerase chain reaction (PCR) pose a direct public health hazard since they do not The presence of A. phagocytophilum DNA in the spleen feed on , their co-occurrence with I. ricinus tissue was tested by a series of nested PCR assays using on the same host can lead to an exchange of patho- specific primers amplifying fragments of rrs (1st PCR: gens among the different tick species [6]. From this ge3a/ge10r; 2nd PCR: ge9f/ge2) [11]. The amplification point of view, small mammals are considered import- was performed as follows: 25 μl reaction mixture ant bridge-hosts and pose an important risk for pub- containing 12.5 μl of Green PCR Master Mix (Rovalab lic health for numerous zoonotic pathogens [2]. GmBH, Teltow, ), 6.5 μl PCR water, 1 μlof Furthermore, rodents are often competent reservoirs each primer (0.01 mM) and 4 μl aliquot of isolated DNA for multi-host pathogens. For instance, mice (Muri- (1 μl of the primary PCR for the nPCR). The amplifica- dae) and voles (Microtidae) are known to be import- tion profile for PCR consisted of 5 min of initial ant reservoirs for zoonotic agents like tick-borne denaturation at 95 °C, followed by 40 cycles of denatur- encephalitis virus (TBEV), Borrelia afzelii,and“Can- ation at 94 °C for 30 s, annealing at 55 °C for 30 s and didatus Neoehrlichia mikurensis” [7]. The role of extension at 72 °C for 60 s, and a final extension at 72 ° small mammals in the epidemiology of A. phagocyto- C for 5 min. For the nPCR, the amplification profile philum in is under debate, previously being consisted of 5 min of initial denaturation at 95 °C, considered important reservoir hosts [8]. Considering followed by 30 cycles of denaturation at 94 °C for 30 s, the high diversity and ubiquity of small mammals and annealing at 55 °C for 30 s and extension at 72 °C for 60 the risk of human contact to their environment s, and a final extension at 72 °C for 5 min. In each PCR (nearly half of Romanian population live and work in reaction set (48 samples), one positive and two negative rural areas and maintain close contact with nature controls were included in order to assess the specificity [9]), the aim of this study was to evaluate the host of the reaction and the possible presence of the presence and genetic diversity of A. phagocytophilum in small cross-contamination. Positive controls consisted of DNA mammals in Romania. extracted from a tick positive for A. phagocytophilum previously confirmed by sequencing [12] and negative controls consisted in sterile water. The PCR was carried Methods out using a T100™ Thermal Cycler (Bio-Rad, London, Small mammals trapping and sampling sites UK). A total of 791 small mammals from 31 species were collected from a variety of habitats in 14 counties in Agarose gel electrophoresis Romania between 2010 and 2015, as previously described PCR products were visualized by electrophoresis in a using snap traps [6]. The trapping of rodents was 1.5% agarose gel stained with SYBR® Safe DNA gel stain performed once per location in different months from late (Invitrogen, Carlsbad, CA, USA) and their molecular spring to early autumn when the vectors are active. In weight was assessed by comparison to a molecular addition, other small mammals which were found dead, marker (O’GeneRuler™ 100 bp DNA Ladder, Thermo were collected from various sources. Whenever needed, Fisher Scientific Inc., Waltham, MA, USA). research permits were obtained from competent author- ities and ethical committees. Each captured or collected DNA sequencing small mammal was identified to species level (according All positive PCR samples were sequenced. PCR products to [10]) and a necropsy was performed. Spleen tissue sam- were purified from amplicons using the QIAquick PCR ples were collected from each . Ticks from the ani- Purification Kit (Qiagen, Hilden, Germany). Sequencing mals were removed and morphologically identified and analysis was performed (Macrogen Europe, Amsterdam, published previously [6]. the ) and the obtained sequences were compared with those available in GenBank™ by Basic DNA extraction Local Alignments Tool (BLAST) analysis. Genomic DNA extraction was performed from the spleen tissue using ISOLATE II Genomic DNA Kit (Bio- Statistical analysis line, London, UK), following the manufacturer’s instruc- Statistical analysis was performed using Epi Info™ 7 tions. For each extraction procedure, negative controls (CDC, Atlanta, GA, USA) software. The total infection were used in order to identify possible cross- prevalence of A. phagocytophilum (95% CI), the infection contamination. DNA from a representative number of prevalence per species and group of species and the samples was quantitatively analyzed using a Nanodrop infection prevalence in each county was assessed using ND-1000 spectrophotometer analyzer (Thermo Fisher the Chi-square independence test. A P-value less than Scientific Inc., Waltham, MA, USA). 0.05 was considered significant. Matei et al. Parasites & Vectors (2018) 11:193 Page 3 of 6

Results CI: 0.05–10.65%) and M. arvalis (1.75% ; 95% CI: 0.04– Twenty out of 791 small mammals were positive for A. 9.39%) (Table 1). No statistically significant differences phagocytophilum DNA presence with an overall preva- in prevalence were observed between different small lence of 2.53% (95% CI: 1.59–3.95%). The small mammal mammal species, nor between taxonomic groups: mice species found positive were: S. araneus (9.09%, 95% CI: (3.06%, 95% CI: 1.61–5.56%), voles (1.14%, 95% CI: 1.92–24.33%), S. cittelus (7.14%, 95% CI: 0.18–33.87%), 0.14–4.07%), (3.94%, 95% CI: 1.28–8.95%) dor- A. flavicollis (6.49%, 95% CI: 2.14–14.51%), A. uralensis mice (1.33%, 95% CI: 0.03–7.21%), or (6.67%, (4.84%, 95% CI: 1.01–13.5%), A. sylvaticus (3.92%, 95% 95% CI: 0.17–31.95%). CI: 0.48–13.46%), M. agrestis (3.85%, 95% CI: 0.10– The positive small mammals originated from Tulcea 19.64%), S. minutus (3.85%, 95% CI: 0.10–19.64%), M. (6/118; 4.84%, 95% CI: 1.80–10.23%), Constanța (6/153; avellanarius (3.13%, 95% CI: 0.08–16.22%), C. suaveolens 3.77%; 95% CI: 1.40–8.03%), Mureş (4/111; 3.48%; 95% (2.44%, 95% CI: 0.06–12.86%), Mus spicilegus (2%, 95% CI: 0.96–8.67%), Cluj (2/217; 0.92%; 95% CI: 0.11–

Table 1 Prevalence of A. phagocytophilum in small mammal species Species Counties Positive/Total Dormouse Glis glis CV 0/43 Muscardinus avellanarius CJ, CV,MS 1/32 Hamster Cricetus cricetus CJ, MS 0/3 Talpa europaea BH, BZ, CJ, CV, MS, SJ, TL 0/14 Mole- Spalax leucodon TL 0/3 Mouse Apodemus agrarius BC, CJ, CT, CV, MS 0/60 Apodemus flavicollis BC, CJ, HR, MS, TL, 5/77 Apodemus sylvaticus CJ, CT, CV, HR, MS, TL 2/51 Apodemus uralensis CT, HR, MS, TL 3/62 Micromys minutus CJ, CT, TL 0/5 Mus musculus AB, BH, CJ, CV, HR, TL 0/55 Mus spicilegus BC, CJ, CT,TL 1/50 Muskrat Ondatra zibethicus BV, TL 0/9 Rat Rattus norvegicus AB, CJ, CT, HR, MS 0/10 Crocidura leucodon CJ, CT, MS, TL 0/21 Crocidura suaveolens CJ, CT, CV, MS, TL 1/41 Neomys anomalus CT, TL 0/3 Neomys fodiens CT, MS 0/2 Sorex alpinus BH 0/1 Sorex araneus AG, CJ, CV, HR, MS 3/33 Sorex minutus CJ, CT, CV, HR, MS, TL 1/26 Sciurus vulgaris BV 0/1 Spermophilus citellus BT, CT, TL 1/14 Vole Arvicola amphibius CT 0/1 Arvicola scherman BH 0/1 Microtus agrestis CJ, HR, TL 1/26 Microtus arvalis BV, CJ, CT, CV, MS, TL 1/57 Microtus subterraneus CJ, HR, MS 0/36 Microtus tatricus HR 0/1 Myodes glareolus CJ, CV, HR, MS 0/53 Bold indicates positive samples, hosts and counties with positive animals detected Abbreviations: AB Alba, AG Argeş, BC Bacău, BT Botoşani, BH Bihor, BV Brașov, BZ Buzău, CJ Cluj, CT Constanţa, CV Covasna, HR Harghita, MS Mureş, SJ Sălaj, TL Tulcea Matei et al. Parasites & Vectors (2018) 11:193 Page 4 of 6

3.29%), Harghita (1/27; 3.57%; 95% CI: 0.09–18.35%) and (5.6%) with that obtained in our study [23]. The A. phago- Covasna (1/124; 0.8%; 95% CI: 0.02–4.38%), without sig- cytophilum prevalence found in M. agrestis, M. arvalis nificance difference between the counties. and S. araneus was lower than in Germany, the UK and The presence of A. phagocytophilum was confirmed by [8, 23, 39]. A higher prevalence in S. araneus sequence analysis with all the sequences (n = 20) show- than in Apodemus spp. was observed in our study, similar ing 99–100% similarity to strains from dogs in Germany with the results from the UK and Switzerland [8, 40]. Our and ticks in and Russia, respectively (GenBank: results have shown no A. phagocytophilum in M. glareo- JX173651, HQ629911, HQ629915). The sequence ana- lus, while in the majority of studies the bank vole is lysis has shown a small degree of variability with only frequently more infected compared with rodents [8, 23, one up to three nucleotides different between the strains 26, 36]. Furthermore, in the studies focused on the bank (Additional file 1). vole, the obtained prevalence ranged between 5 and 22% [14, 28]. The differences in prevalence between different Discussion studies could be explained by several factors such as abun- The aim of present study was to evaluate the host dance and population structure of the tick vector and the spectrum and prevalence of A. phagocytophilum in small abundance and diversity of potential reservoir hosts, both mammal species across Romania. The results have being influenced by the climatic and ecological features shown an overall low prevalence, with no significant including sampling period. Others factors which may in- difference between host species and geographical areas. fluence the prevalence are related to the type and quality The low genetic diversity and the large number of posi- of samples and methods used. Conservative strategies are tive species from our study confirm the low host specifi- usually used for screening based on rrs and groEL genes city of A. phagocytophilum observed at least for some or multicopy of the major surface proteins such as msp2 variants [13]. and msp4 (reviewed in [41]). Among different types of Anaplasma phagocytophilum DNA was previously samples, our previous research and literature data suggest detected in several small mammal species such as A. spleen tissue as the most suitable organ for the detection agrarius, A. flavicollis, A. uralensis, A. sylvaticus, M. of A. phagocytophilum [41]. musculus, M. agrestis, M. arvalis, M. oeconomus, Myodes Small mammals were previously considered reservoir glareolus, S. araneus and C. russula in several European hosts for A. phagocytophilum [8]. They are important countries [8, 13–29]. There are also several reports on hosts for immature stages of I. ricinus [42], and also for the occurrence of A. phagocytophilum infection in Eri- I. trianguliceps [38] and I. hexagonus [30], which may naceus europaeus [30–32], E. roumanicus [33] and Rat- transmit the infection. However, since most rodents are tus rattus [15] and in large rodents such as Hystrix short-lived animals and that the infection with A. phago- cristata [34]. In addition to small mammals, A. phagocy- cytophilum seems to be transient [30], their role as suit- tophilum was detected in a large variety of hosts includ- able reservoir hosts is currently under debate. Moreover, ing birds, domestic and wild carnivores, livestock, wild in recent years it has been suggested that small mam- ruminants, wild boars and humans (reviewed in [13]). mals and their ticks are involved in a separate enzootic To our knowledge, this is the first report of A. phagocy- cycle. This hypothesis is sustained by the phylogenetic tophilum in S. minutus, C. suaveolens, M. spicilegus, M. analysis on ankA and groEL genes which showed that avellanarius and S. citellus. strains isolated from small mammals differ genetically The overall prevalence in small mammals in the from those circulating in I. ricinus ticks, domestic rumi- present study was within the prevalence intervals from nants, wild boar, dogs, horses or humans [26, 28, 39, 43]. other reports (reviewed in [13]). The A. phagocytophilum It has been suggested that I. trianguliceps might be the prevalence recorded in other European countries present vector of these rodent strains in the UK [39, 40]. a high variability especially for several species such as: A. Furthermore, in Switzerland and Slovakia, A. phagocyto- agrarius (1.28–13.54%) [24, 35], A. flavicollis (0.48–18%) philum was not detected in I. ricinus ticks feeding on [22, 36], A. sylvaticus (0.61–11.1%) [19, 20], M. agrestis rodents even though A. phagocytophilum was detected (0.28–25%) [37, 38], M. arvalis (0.28–25%) [29, 37] and in questing I. ricinus in the same areas [44]orinI. trian- M. glareolus (0.30–21.85%) [25, 28]. In the present study, guliceps removed from the same hosts [26]. Moreover, prevalence of A. phagocytophilum in A. flavicollis none of the artificially-fed I. ricinus became infected (6.49%) was lower than in the [16] and after engorging blood from positive rodents [44]. How- higher than in Germany and Switzerland [8, 22]. Apode- ever, more experimental studies, including xenodiagnos- mus sylvaticus from Romania was less infected than A. fla- tics are needed to confirm this separate enzootic cycle. vicollis, in contrast with results obtained in Switzerland Although the sequence analysis confirmed the presence [8]. Anaplasma phagocytophilum was previously found in of A. phagocytophilum DNA in small mammal species, one A. uralensis in Slovakia, having a similar prevalence experimental studies are required to demonstrate the Matei et al. Parasites & Vectors (2018) 11:193 Page 5 of 6

role of these species as competent host. Moreover, the rrs Competing interests gene is highly conservative and further research on other The authors declare that they have no competing interests. genes or by more sensitive techniques such as multilocus sequence typing (MLST) or multilocus variable-number Publisher’sNote tandem-repeat analysis (MLVA), is needed in order to Springer Nature remains neutral with regard to jurisdictional claims in properly characterize these strains present in small mam- published maps and institutional affiliations. mals in Romania. Based on the data obtained in this study Author details it is unclear if these strains are hazardous to humans or 1Department of Parasitology and Parasitic Diseases, Faculty of Veterinary domestic animals and if they are transmitted by public Medicine, University of Agricultural Sciences and Veterinary Medicine, 2 health relevant tick species. Cluj-Napoca, Romania. Department of Microbiology, Immunology and Epidemiology, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania. 3Department of Conclusions Reproduction, Obstetrics and Pathology of Animal Reproduction, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Several small mammal species are described in the lit- Medicine, Cluj-Napoca, Romania. erature as carriers of A. phagocytophilum. Besides these, our results have shown other species such as S. minutus, Received: 4 January 2018 Accepted: 8 March 2018 C. suaveolens, M. spicilegus, M. avellanarius and S. citel- lus, which can also be infected with A. phagocytophilum. However, it is unclear whether these animals can act as References 1. Durden LA. , host associations, life cycles and vectorial competent reservoir hosts and for which A. phagocyto- importance of ticks parasitizing small mammals. In: Morand S, Krasnov BR, philum variants. Poulin R, editors. Micromammals and macroparasites: From evolutionary ecology to management. Tokyo: Springer-Verlag; 2006. p. 91–102. 2. Gratz N. Vector-and rodent-borne diseases in Europe and North America: Additional files Distribution, public health burden, and control. Cambridge: Cambridge University Press; 2006. Additional file 1: Alignment of the sequences obtained in this study. 3. Gage KL, Kosoy MY. Non-commensal rodents and lagomorphs. In: Bonnefoy Nucleotides different between the strains are marked with different X, Kampen H, Sweeney K, editors. Public health significance of urban pests. colors. (DOCX 19 kb) Copenhagen: World Health Organization; 2008. p. 421–76. 4. Heyman P, Cochez C, Hofhuis A, Van Der Giessen J, Sprong H, Porter SR, et al. A clear and present danger: tick-borne diseases in Europe. Expert Rev Abbreviations Anti-Infect Ther. 2010;8(1):33–50. BLAST: Basic Local Alignments Tool; MLST: Multilocus sequence typing; 5. Mihalca AD, Sándor AD. The role of rodents in the ecology of MLVA: Multilocus variable-number tandem-repeat analysis; nPCR: Nested PCR; and associated pathogens in central and eastern Europe. Front Cell Infect rrs: 16S rRNA Microbiol. 2013;3:56. 6. Mihalca AD, Dumitrache MO, Sándor AD, Magdaş C, Oltean M, Györke A, et Acknowledgments al. Tick parasites of rodents in Romania: host preferences, community The abstract of this paper has been presented at a local student symposium structure and geographical distribution. Parasit Vectors. 2012;5:266. by an undergraduate student, Emese Beáta Tamás, to whom we want to 7. Rizzoli A, Silaghi C, Obiegala A, Rudolf I, Hubálek Z, Földvári G, et al. Ixodes thank for her contribution. ricinus and its transmitted pathogens in urban and peri-urban areas in Europe: new hazards and relevance for public health. Front Public Health. Funding 2014;2:251. The research was supported from grant PN-II-RU-TE-2014-4-0919: TE/298/ 8. Liz JS, Anderes L, Sumner JW, Massung RF, Gern L, Rutti B, Brossard M. PCR 2015. This paper was published under the frame of EurNegVec COST Action detection of granulocytic ehrlichiae in Ixodes ricinus ticks and wild small – TD1303. mammals in western Switzerland. J Clin Microbiol. 2000;38(3):1002 7. 9. Vincze M, Kerekes K. Impact of CAP’s pillars on Romanian rural employment. In: Proceedings of the Aspects and Visions of Applied Economics and Availability of data and materials Informatics Conference, Debrecen, 2009;4:68–73. The data supporting the conclusions of this article are included within the 10. Mitchell-Jones AJ, Moutou F, Zima J. Mammals of Europe, North Africa and article. Raw data used and/or analyzed during the current study are available the Middle East. London: A&C Black; 2009. from the corresponding author upon reasonable request. 11. Massung RF, Slater K, Owens JH, Nicholson WL, Mather TN, Solberg VB, Olson JG. Nested PCR assay for detection of granulocytic ehrlichiae. J Clin ’ Authors contributions Microbiol. 1998;36(4):1090–5. GDA, ADS, NF, LB, CMG and ADM collected the small mammals. IAM and 12. Matei IA, Kalmár Z, LupşeM,D’Amico G, Ionică AM, Dumitrache MO, et al. AMI performed the necropsy. IAM and ZK performed DNA extraction. IAM The risk of exposure to rickettsial infections and human granulocytic conducted DNA detection by PCR. IAM and AC performed agar gel anaplasmosis associated with Ixodes ricinus tick bites in humans in Romania: electrophoresis. IAM analyzed and interpreted statistical analysis and wrote a multiannual study. Ticks Tick Borne Dis. 2017;8(3):375–8. the manuscript. GDA and ADM were major contributors in writing the 13. Stuen S, Granquist EG, Silaghi C. Anaplasma phagocytophilum - a manuscript. All authors read and approved the final manuscript. widespread multi-host pathogen with highly adaptive strategies. Front Cell Infect Microbiol. 2013;3:31. Ethics approval 14. Bown KJ, Begon M, Bennett M, Woldehiwet Z, Ogden NH. Seasonal This study was approved by the USAMV CN Bioethics Committee with the dynamics of Anaplasma phagocytophila in a rodent-tick (Ixodes trianguliceps) registration number 23/ 21-09-2015, following the EU 2010/63 and National system, . Emerg Infect Dis. 2003;9(1):63–70. directives Ord. 28/31-08-2011 and National Law 206/2004. 15. Christova I, Gladnishka T. Prevalence of infection with Francisella tularensis, Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in rodents Consent for publication from an endemic focus of tularemia in . Ann Agric Environ Med. Not applicable. 2004;12(1):149–52. Matei et al. Parasites & Vectors (2018) 11:193 Page 6 of 6

16. Hulínská D, Langrová K, Pejčoch M, Pavlasek I. Detection of Anaplasma 38. Bown KJ, Begon M, Bennett M, Birtles RJ, Burthe S, Lambin X, et al. Sympatric phagocytophilum in animals by real-time polymerase chain reaction. APMIS. Ixodes trianguliceps and Ixodes ricinus ticks feeding on field voles (Microtus 2004;112(4–5):239–47. agrestis): potential for increased risk of Anaplasma phagocytophilum in the 17. Smetanová K, Schwarzová K, Kocianová E. Detection of Anaplasma United Kingdom? Vector Borne Zoonotic Dis. 2006;6:404–10. phagocytophilum, Coxiella burnetii, Rickettsia spp., and Borrelia burgdorferi s.l. 39. Bown KJ, Lambin X, Telford GR, Ogden NH, Telfer S, Woldehiwet Z, Birtles in Ticks, and wild-living animals in Western and Middle Slovakia. Ann N Y RJ. Relative importance of Ixodes ricinus and Ixodes trianguliceps as vectors Acad Sci. 2006;1078(1):312–5. for Anaplasma phagocytophilum and Babesia microti in field vole (Microtus 18. Grzeszczuk A, Karbowiak G, Ziarko S, Kovalchuk O. The root-vole Microtus agrestis) populations. Appl Environ Microbiol. 2008;74(23):7118–25. oeconomus (Pallas, 1776): a new potential reservoir of Anaplasma 40. Bown KJ, Lambin X, Ogden NH, Begon M, Telford G, Woldehiwet Z, Birtles phagocytophilum. Vector Borne Zoonotic Dis. 2006;6(3):240–3. RJ. Delineating Anaplasma phagocytophilum ecotypes in coexisting, discrete 19. Barandika JF, Hurtado A, García-Esteban C, Gil H, Escudero R, Barral M, et al. enzootic cycles. Emerg Infect Dis. 2009;15(12):1948–54. Tick-borne zoonotic bacteria in wild and domestic small mammals in 41. Silaghi C, Santos AS, Gomes J, Christova I, Matei IA, Walder G, et al. northern . Appl Environ Microbiol. 2007;73(19):6166–71. Guidelines for the direct detection of Anaplasma spp. in diagnosis and 20. Bray DP, Bown KJ, Stockley P, Hurst JL, Bennett M, Birtles RJ. Haemoparasites epidemiological studies. Vector Borne Zoonotic Dis. 2017;17(1):12–22. of common shrews (Sorex araneus) in northwest England. Parasitology. 42. Nilsson A. Seasonal occurrence of Ixodes ricinus () in vegetation and on 2007;134(06):819–26. small mammals in southern . Ecography. 1988;11(3):161–5. 21. Matsumoto K, Joncour G, Lamanda P, Inokuma H, Brouqui P. Detection of 43. Majazki J, Wüppenhorst N, Hartelt K, Birtles R, Von Loewenich FD. Anaplasma phagocytophilum and Ehrlichia sp. HF strains in Ixodes ricinus Anaplasma phagocytophilum strains from voles and shrews exhibit specific ticks in Brittany, . Clin Microbiol Infect. 2007;13(3):338–41. ankA gene sequences. BMC Vet Res. 2013;9(1):235. 22. Hartelt K, Pluta S, Oehme R, Kimmig P. Spread of ticks and tick-borne diseases 44. Burri C, Schumann O, Schumann C, Gern L. Are Apodemus spp. mice and in Germany due to global warming. Parasitol Res. 2008;103(1):109–16. Myodes glareolus reservoirs for Borrelia miyamotoi, “Candidatus Neoehrlichia 23. Štefančíková A, Derdáková M, Lenčáková D, Ivanová R, Stanko M, Čisláková mikurensis”, Rickettsia helvetica, R. monacensis and Anaplasma L, Peťko B. Serological and molecular detection of Borrelia burgdorferi sensu phagocytophilum? Ticks Tick Borne Dis. 2014;5(3):245–51. lato and Anaplasmataceae in rodents. Folia Microbiol. 2008;53(6):493–9. 24. Krücken J, Schreiber C, Maaz D, Kohn M, Demeler J, Beck S, et al. A novel high-resolution melt PCR assay discriminates Anaplasma phagocytophilum and “Candidatus Neoehrlichia mikurensis”. J Clin Microbiol. 2013;51:1958–61. 25. Baráková I, Derdáková M, Carpi G, Rosso F, Collini M, Tagliapietra V, et al. Genetic and ecologic variability among Anaplasma phagocytophilum strains, northern Italy. Emerg Infect Dis. 2014;20(6):1082–5. 26. Blaňarová L, Stanko M, Carpi G, Miklisová D, Víchová B, Mošanský L, et al. Distinct Anaplasma phagocytophilum genotypes associated with Ixodes trianguliceps ticks and rodents in central Europe. Ticks Tick Borne Dis. 2014; 5(6):928–38. 27. Jahfari S, Coipan EC, Fonville M, Van Leeuwen AD, Hengeveld P, Heylen D, et al. Circulation of four Anaplasma phagocytophilum ecotypes in Europe. Parasit Vectors. 2014;7:365. 28. Kallio ER, Begon M, Birtles RJ, Bown KJ, Koskela E, Mappes T, Watts PC. First report of Anaplasma phagocytophilum and Babesia microti in rodents in Finland. Vector-Borne Zoonotic Dis. 2014;14(6):389–93. 29. Szekeres S, Coipan EC, Rigó K, Majoros G, Jahfari S, Sprong H, Földvári G. “Candidatus Neoehrlichia mikurensis” and Anaplasma phagocytophilum in natural rodent and tick communities in Southern Hungary. Ticks Tick Borne Dis. 2015;6(2):111–6. 30. Skuballa J, Petney T, Pfäffle M, Taraschewski H. Molecular detection of Anaplasma phagocytophilum in the European (Erinaceus europaeus) and its ticks. Vector-Borne Zoonotic Dis. 2010;10(10):1055–7. 31. Silaghi C, Skuballa J, Thiel C, Pfister K, Petney T, Pfäffle M, et al. The European hedgehog (Erinaceus europaeus) - a suitable reservoir for variants of Anaplasma phagocytophilum? Ticks Tick Borne Dis. 2012;3(1):49–54. 32. Huhn C, Winter C, Wolfsperger T, Wüppenhorst N, Smrdel KS, Skuballa J, et al. Analysis of the population structure of Anaplasma phagocytophilum using multilocus sequence typing. PLoS One. 2014; 9(4):e93725. 33. Földvári G, Rigó K, Jablonszky M, Biró N, Majoros G, Molnár V, Tóth M. Ticks and the city: ectoparasites of the Northern white-breasted hedgehog (Erinaceus roumanicus) in an urban park. Ticks Tick Borne Dis. 2011;2(4):231–4. 34. Torina A, Alongi A, Naranjo V, Estrada-Peña A, Vicente J, Scimeca S, et al. Submit your next manuscript to BioMed Central Prevalence and genotypes of Anaplasma species and habitat suitability for and we will help you at every step: ticks in a Mediterranean ecosystem. Appl Environ Microbiol. 2008;74(24): 7578–84. • We accept pre-submission inquiries 35. Christova I, Dimitrov H, Trifonova I, Gladnishka T, Mitkovska V, Stojanova A, • Our selector tool helps you to find the most relevant journal et al. Detection of human tick-borne pathogens in rodents from Bulgaria. • Acta Zool Bulg. 2012;64(Suppl. 4):111–4. We provide round the clock customer support 36. Chastagner A, Moinet M, Perez G, Roy E, McCoy KD, Plantard O, et al. • Convenient online submission Prevalence of Anaplasma phagocytophilum in small rodents in France. Ticks • Thorough peer review Tick Borne Dis. 2016;7(5):988–91. • Inclusion in PubMed and all major indexing services 37. Obiegala A, Pfeffer M, Pfister K, Tiedemann T, Thiel C, Balling A, et al. “Candidatus Neoehrlichia mikurensis” and Anaplasma phagocytophilum: • Maximum visibility for your research prevalences and investigations on a new transmission path in small mammals and ixodid ticks. Parasit Vectors. 2014;7:563. Submit your manuscript at www.biomedcentral.com/submit